Embryonic retinal stem cells give rise to the diverse assortment of neuronal and glial cells, which form the mature eye. In retinal injury and blinding diseases, such as glaucoma and retinitis pigmentosa, retinal cells are lost and never regenerate. Replacement of damaged retinal cells with multipotent retinal stem (RS) cells has been hampered by an inability to isolate and culture sufficient quantities of these rare cells. Once the intrinsic and extrinsic cues regulating their generation and differentiation are identified, retinal stem cells will become a precious resource for cell replacement therapies. Molecular and genetic evidence shows that embryonic RS cells are specified in the anterior neural plate by the combined action of eye field transcription factors (ET, Rx, Pax6, Six3, Lhx2, til and Optx2). Following RS cell specification, additional extrinsic and intrinsic cues direct their differentiation into mature cell types in the eye. We recently discovered that co-expression of the eye field transcription factors is sufficient to induce RS cells and fully functional eyes at high frequency in the frog embryo. In the frog Xenopus laevis, RS cells develop at room temperature in a simple salt solution in just hours. Modern molecular and genetic techniques make it possible to manipulate and monitor gene expression in living embryos at virtually any developmental stage. The frog eye shares common molecular, developmental, structural, and functional processeswith other vertebrate species, including humans. These strengths make Xenopus a uniquely suited system to analyze RS cells and eye development. RNA blastomere injections will be used to overexpress cocktails of wild-type with function blocking forms of eye field transcription factors (EFTFs) in developing embryos. These experiments will determine the minimum number of EFTFs necessary and sufficient to generate multipotent RS cells and ectopic eyes in vivo. (Aim 1). Next, cultured RS cells will be used to determine the combination of extrinsic and intrinsic factors regulating their proliferation and differentiation (Aim 2). Finally, we will transplant in vitro generated RS cells into the embryo to determine if they differentiate, integrateand survive in the mature retina (Aim 3). Together, these experimentswill identify the effectors regulating RS cell specification, proliferation and differentiation and test the ability of in vitro generated RS cells to repopulate the mature retina. These studies will advance our basic understanding of the underlying mechanisms regulating retinal development, and provide the fundamental work necessary for the ultimate goal of using RS cells to treat blinding diseases.